The Toxic Effects of Cigarette Additives. Philip Morris' Project Mix Reconsidered: An Analysis of Documents Released through Litigation
Background:
In 2009, the promulgation of US Food and Drug Administration (FDA) tobacco regulation focused attention on cigarette flavor additives. The tobacco industry had prepared for this eventuality by initiating a research program focusing on additive toxicity. The objective of this study was to analyze Philip Morris' Project MIX as a case study of tobacco industry scientific research being positioned strategically to prevent anticipated tobacco control regulations.
Methods and Findings:
We analyzed previously secret tobacco industry documents to identify internal strategies for research on cigarette additives and reanalyzed tobacco industry peer-reviewed published results of this research. We focused on the key group of studies conducted by Phillip Morris in a coordinated effort known as “Project MIX.” Documents showed that Project MIX subsumed the study of various combinations of 333 cigarette additives. In addition to multiple internal reports, this work also led to four peer-reviewed publications (published in 2001). These papers concluded that there was no evidence of substantial toxicity attributable to the cigarette additives studied. Internal documents revealed post hoc changes in analytical protocols after initial statistical findings indicated an additive-associated increase in cigarette toxicity as well as increased total particulate matter (TPM) concentrations in additive-modified cigarette smoke. By expressing the data adjusted by TPM concentration, the published papers obscured this underlying toxicity and particulate increase. The animal toxicology results were based on a small number of rats in each experiment, raising the possibility that the failure to detect statistically significant changes in the end points was due to underpowering the experiments rather than lack of a real effect.
Conclusion:
The case study of Project MIX shows tobacco industry scientific research on the use of cigarette additives cannot be taken at face value. The results demonstrate that toxins in cigarette smoke increase substantially when additives are put in cigarettes, including the level of TPM. In particular, regulatory authorities, including the FDA and similar agencies elsewhere, could use the Project MIX data to eliminate the use of these 333 additives (including menthol) from cigarettes.
: Please see later in the article for the Editors' Summary
Published in the journal:
The Toxic Effects of Cigarette Additives. Philip Morris' Project Mix Reconsidered: An Analysis of Documents Released through Litigation. PLoS Med 8(12): e32767. doi:10.1371/journal.pmed.1001145
Category:
Research Article
doi:
https://doi.org/10.1371/journal.pmed.1001145
Summary
Background:
In 2009, the promulgation of US Food and Drug Administration (FDA) tobacco regulation focused attention on cigarette flavor additives. The tobacco industry had prepared for this eventuality by initiating a research program focusing on additive toxicity. The objective of this study was to analyze Philip Morris' Project MIX as a case study of tobacco industry scientific research being positioned strategically to prevent anticipated tobacco control regulations.
Methods and Findings:
We analyzed previously secret tobacco industry documents to identify internal strategies for research on cigarette additives and reanalyzed tobacco industry peer-reviewed published results of this research. We focused on the key group of studies conducted by Phillip Morris in a coordinated effort known as “Project MIX.” Documents showed that Project MIX subsumed the study of various combinations of 333 cigarette additives. In addition to multiple internal reports, this work also led to four peer-reviewed publications (published in 2001). These papers concluded that there was no evidence of substantial toxicity attributable to the cigarette additives studied. Internal documents revealed post hoc changes in analytical protocols after initial statistical findings indicated an additive-associated increase in cigarette toxicity as well as increased total particulate matter (TPM) concentrations in additive-modified cigarette smoke. By expressing the data adjusted by TPM concentration, the published papers obscured this underlying toxicity and particulate increase. The animal toxicology results were based on a small number of rats in each experiment, raising the possibility that the failure to detect statistically significant changes in the end points was due to underpowering the experiments rather than lack of a real effect.
Conclusion:
The case study of Project MIX shows tobacco industry scientific research on the use of cigarette additives cannot be taken at face value. The results demonstrate that toxins in cigarette smoke increase substantially when additives are put in cigarettes, including the level of TPM. In particular, regulatory authorities, including the FDA and similar agencies elsewhere, could use the Project MIX data to eliminate the use of these 333 additives (including menthol) from cigarettes.
: Please see later in the article for the Editors' Summary
Introduction
The tobacco industry has been preparing for regulation of its products since at least 1963 when Philip Morris (PM) Vice President of Research and Development Helmut Wakeham expressed concern that the existing US Food and Drug Administration (FDA) “generally recognized as safe (GRAS)” for food additives would not apply to cigarettes and observed that, “Strictly speaking, only published studies could be used to establish a GRAS list for things which are inhaled. Relatively few such studies have been made. This concept would provide a rather formidable barrier in the case of cigarettes” [1]. In 1984 the safety of cigarette additives first became a public issue in the United States [2]. Additives are important elements of tobacco products because they allow manufacturers to modify the sensory and pharmacological properties of tobacco products in a way that affects initiation and cessation (with additives that make the smoke less harsh or more pleasant to the user) or bioavailability and impact of nicotine (through changes in smoke pH or through additives such as menthol) (Box 1) [3],[4]. Regulating additives, including prohibiting their use, poses a serious threat to the tobacco companies. The threat of regulation grew in 1995 when President William Clinton allowed the FDA to regulate cigarettes. While the industry won a Supreme Court ruling that the FDA did not have authority to regulate tobacco products, PM executives recognized by the mid-1990s that regulation was inevitable. They initiated efforts to shape the legislation that would become the 2009 Family Smoking Prevention and Tobacco Control Act [5] granting the FDA authority over tobacco products [6]–[8]. (Among the first actions taken by the newly established FDA Center for Tobacco Products in 2009 was prohibition of flavor additives in cigarettes, with the exceptions of tobacco and menthol [9].) While US legislation over tobacco product regulation was being debated, several tobacco companies developed procedures for assessing the biological effects of new products that delivered nicotine differently than conventional cigarettes [10]. In addition, tobacco product regulation became a global issue with development and implementation of the 2005 World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) [11], whose articles 9, 10, and 11 require parties to regulate tobacco products.
Box 1. Excerpt from FCTC implementing guidelines on ingredients (articles 9 and 10) [3, Section 3.1.2.2]
Ingredients used to increase palatability
The harsh and irritating character of tobacco smoke provides a significant barrier to experimentation and initial use. Tobacco industry documents have shown that significant effort has been put into mitigating these unfavourable characteristics. Harshness can be reduced in a variety of ways including: adding various ingredients, eliminating substances with known irritant properties, balancing irritation alongside other significant sensory effects, or altering the chemical properties of tobacco product emissions by adding or removing specific substances.
Some tobacco products contain added sugars and sweeteners. High sugar content improves the palatability of tobacco products to tobacco users. Examples of sugars and sweeteners used in these products include glucose, molasses, honey, and sorbitol.
Masking tobacco smoke harshness with flavours contributes to promoting and sustaining tobacco use. Examples of flavouring substances include benzaldehyde, maltol, menthol, and vanillin.
Spices and herbs can also be used to improve the palatability of tobacco products. Examples include cinnamon, ginger, and mint.
Recommendation:
Parties should regulate, by prohibiting or restricting, ingredients that may be used to increase palatability in tobacco products.
Ingredients indispensable for the manufacturing of tobacco products and not linked to attractiveness should be subject to regulation according to national law.
In addition to its political response [6],[7], PM USA reorganized its internal scientific activities to respond to product regulation. In a July 1997 memo “1997 FDA Compliance Preparation,” PM's Vice President of Research and Development stated that, “It is important that we quickly … move to prepare ourselves for these potential regulations” [12]. PM had been analyzing smoke constituents and performing toxicology testing on cigarettes at its European laboratories for decades [13]. In August 1997 PM began to plan and conduct studies of the chemical and toxicological effects of 333 cigarette additives through its “Project MIX” [14]. Project MIX included chemical analysis of smoke, in vitro mutagenicity and cytotoxicity testing, and in vivo rat inhalation toxicology studies. The studies resulted in four papers published together in Food and Chemical Toxicology in January 2002 [15]–[18]. The overall conclusion reported by PM in these four papers was that, “The statistically significant changes detected in some of the parameters measured in these studies were considered incidental, without influence on the overall biological effects normally seen with cigarette smoke exposure. There was no indication of any new effects that could be attributable to ingredients” [15].
The use of cigarette additives is an important concern of the FDA. Among the first actions taken by the newly established FDA Center for Tobacco Products in 2009 was prohibition of flavor additives in cigarettes, with the exceptions of tobacco and menthol [9], and in 2011 the FDA's Tobacco Products Scientific Advisory Committee concluded that, “Removal of menthol cigarettes from the marketplace would benefit public health in the United States” [19].
PM has used the published Project MIX papers to assert the safety of individual additives, citing the not yet published papers in a 2001 series of white papers. These papers include Evaluation of Menthol for Use as a Cigarette Ingredient [20] and at least seven others (cocoa [21], propylene glycol [22], vanilla extract [23], glycerol [24], sweet orange oil [25], and licorice extract [26]). Lorillard scientist, J. Daniel Heck, a member of the FDA Tobacco Products Scientific Advisory Committee [27] cited Project MIX findings in a 2010 review paper arguing that use of menthol in cigarettes is safe [28].
We used documents made public as a result of litigation against the tobacco industry to investigate the origins and design of Project MIX and conducted additional analyses of the results. This assessment raises questions about the four papers' conclusion that the results “did not demonstrate any meaningful effect of these [333] ingredients on the toxicity of cigarettes” [15].
Methods
Tobacco Industry Documents
We systematically examined tobacco industry documents in the University of California San Francisco Legacy Tobacco Documents Library (LTDL; http://legacy.library.ucsf.edu/), which were discovered using established protocols and methods of searching documents based on the snowball technique, by which previous searches inform subsequent searches [29]–[31]. In order to address our research question about how the tobacco industry uses scientific research as a strategy to oppose anticipated tobacco control policy, we began with the search terms “ingredients,” “additives,” and “toxicity.” This initial search yielded thousands of documents, sorted for relevance by the optical character recognition capability of LTDL on the basis of the number of times a search term appears in a document. Screening the documents qualitatively in batches of 50 to 100 helped refine and narrow the search allowing us to collect documents that would help construct a history and provide context for the information that was coming forth. Additional relevant documents were returned by examining adjacent documents (Bates numbers), and searching for key individuals mentioned and locations in which relevant documents were found. The iterative process of searching, analyzing, and refining led to the identification of Project MIX as a key search term. We reviewed approximately 500 relevant documents in detail, including German language documents from the PM-owned European laboratories where Project MIX was conducted. Our analysis is necessarily limited only to those materials (about 60 million pages at the time we conducted this study) that are available as a result of litigation [29].
Results
Design Elements
Origins of Project MIX
The tobacco companies have a long history of animal research, dating back to at least the 1960s, when they attempted to identify and eliminate the chemicals that caused cancer as part of the effort to develop a “safe cigarette” (see in [32], Chapter 4). Beginning in the late 1970s, PM scientists conducted mouse inhalation and skin painting studies to assess potential cigarette additives, including urea and carmel [33], cocoa [34], and glycerol [35]. In the 1980s PM routinely performed chemical analysis of smoke [36],[37], cytotoxicity testing (Ames and neutral red uptake tests) for mutagenicity [38],[39], and in vivo rodent toxicology studies [40]–[42], honing the techniques for performing these assays at their Institut für Biologische Forschung (INBIFO) in Germany and Contract Research Center (CRC) in Belgium [13].
In March 1994 lawyers representing PM (at the Covington & Burling law firm) commissioned a report on cigarette additives written by six toxicology experts that concluded “ingredients are not hazardous under the conditions of use” [43]. A list of 599 ingredients used in the manufacture of cigarettes was made public for the first time in August 1994 [44] that the industry regarded as “safe” [45]. By 1997 PM joined the Society of Toxicology and the American College of Toxicology to position PM scientists to present and publish results of the INBIFO trials and the six toxicology experts [46].
PM scientists designed Project MIX in 1997 to evaluate the effects of cigarette additives on smoke chemistry, in vitro mutagenicity and cytotoxicity, and in vivo biological activity [14]. In 2001 the four papers written by PM scientists based on the Project MIX results were accepted for publication in Food and Chemical Toxicology [15]–[18] through a process that PM scientist and leader of Project MIX Edward Carmines described to coworkers as “an inside job. We went to a journal whose editor knew us” [47].
Carmines' comment is well founded. The then editor of Food and Chemical Toxicology, Joseph Borzelleca, was a member of the US tobacco industry's Council for Tobacco Research Scientific Advisory Board [48] and PM Scientific Advisory Board [49] and had a long history of doing contract research and consulting for PM (e.g., see [50] and [51]; there are thousands of documents mentioning Borzelleca in LTDL). The associate editor, P.J. van Bladeren, was coauthor of a paper at the 1991 meeting sponsored by Indoor Air International, a group managed by tobacco industry lawyers, “International Conference: Priorities for Indoor Air Research and Action” [52] that served as the launch for a nominally peer-reviewed journal that could be used to publish research supporting the tobacco industry's position on secondhand smoke [53]. Susan Barlow, one of two review editors, coauthored a PM-funded review paper that, after incorporating comments from PM, questioned the evidence linking secondhand smoke and sudden infant death syndrome [54]. Eleven of the journal's International Editorial Board members had ties to the tobacco industry: three were employees (A.W. Hayes, D.J. Doolittle [55]–[57], and Y.P. Dragan [58]–[60]; two held positions on PM Scientific Advisory Board (M. Pariza and S.L. Taylor [61]); and six others had tobacco industry funding or other connections (O.I. Aruoma [62],[63], A.R. Boobis [64]–[66], H.R. Glatt [67]–[69], G.C. Hard [70],[71], B. Pool-Zobel [72],[73],and H. Poulsen [74]–[77]).
Additive selection
Project MIX examined the potential chemical and biological effects of 333 additives used in cigarette manufacturing, selected because they “are representative of flavors used throughout the world by Philip Morris” [78]. The 333 additives represent some of the 599 that had been reported to be added in cigarette manufacturing [44].
The 333 additives selected for testing under Project MIX were divided into three overlapping “ingredient groups” [15]. The additives were added to tobacco during the cigarette manufacturing process to produce the test cigarettes. The control cigarette contained tobacco only. Two target levels for each additive, a low level that “approximated the level considered to be reflective of those used in commercial cigarettes” [15] and a high level, 1.5 or 3 times multiple of the low level. (The exception was menthol in ingredient group 3, which was only studied at a single level, 18,000 parts per million [ppm], in both the low and high groups “because no more material could be physically incorporated into the test cigarette” [15].) Carmines, et al. state in the published paper [15] that they could only vary the levels by a factor of 1.5–3 because it was the maximum amount of the ingredients that the cigarettes would hold and still “burn in manner similar to the control cigarette.” Tobacco was removed from the test cigarettes to keep the cigarette mass constant.
The specific contents of the test cigarettes are listed in tabular form in the published paper [15] and can be summarized as follows: ingredient group 1, 177 additives; ingredient group 2, 277 additives (including 117 also in group 1); ingredient group 3, mostly menthol plus cocoa shells, licorice extract, and corn syrup.
The process or logic for assigning additives to groups is not evident from the industry documents or published papers. It is not clear how or why PM selected these 333 additives and the specific amounts or combinations used, nor why the other 266 were not selected for testing in Project MIX. The published paper [15] simply states that the additives are “representative of ingredients [additives] used throughout the world,” are “food-grade quality or better,” and were added during the test cigarette manufacturing process “to closely mimic the actual process” that “were constructed to resemble typical commercial blended cigarettes.”
To prepare for Project MIX, PM USA manufactured seven test cigarettes under these product codes: 97.MG.292 for control, 97.MG.295 and 97.MG.296 for ingredient group 1 low and high, 97.MJ.125 and 97.MJ.126 for ingredient group 2 low and high, and 97.MJ.127 and 97.MJ.128 for ingredient group 3 low and high [79]. The test cigarettes were released to INBIFO in January 1998 by PM's US Product Testing Laboratory scientist after performance tests were completed [80].
Chemical analysis of the smoke
INBIFO measured 51 mainstream smoke constituents in the Project MIX test cigarettes. According to Carmines, these constituents were selected from lists prepared by the US Consumer Product Safety Commission (CPSC) [81] and International Agency for Research on Cancer [16] (IARC) monograph 38 [82]. A 1997 memo written by PM scientist Rick Solana [83],[84] indicates that PM had created a list of 39 as a “current smoke constituent chemistry list for analyzing mainstream smoke in product integrity testing” on the basis of the IARC and CPSC lists screened against the 1994 National Toxicology Program (NTP) carcinogen list.
We could not determine how the 51 constituents were selected for use in Project MIX. The published paper from Project MIX [16] acknowledges the selection was “to a certain degree subjective and not based on extensive risk assessments,” and argues that some analytes were not included because “there is no straightforward toxicological interpretation for changes that might occur” or because “their method development is still in progress” [16]. Combining the CPSC and IARC lists yields 118 compounds (Text S1, Table S-1). Solana noted (in 1997, of the 39 constituent list), that “polyaromatic hydrocarbons…will be kept on the current list this is due to the focus on this class of compounds. Even these compounds, however, might be considered for discontinuing should they prove to consistently track the polyaromatic hydrocarbons which are on the recommended list” [83]. This decision could account for the omission of some constituents from Project MIX.
The list of 72 constituents not measured in Project MIX includes 11 polycyclic aromatic hydrocarbons (PAHs). PAHs are of particular concern because they cause carcinogenic and noncarcinogenic disease in animals and in humans [85]–[87]. Project MIX also included eight chemicals not on the CPSC, IARC, or National Toxicology Program (NTP) lists: three PAHs: acenaphthene, acenaphthylene, benzo[ghi]perylene; naphthalene; m-, o-, and p-cresol; and particle size distribution. One of these PAHs was also identified in a single ingredient smoke analysis study, conducted by PM under the code name URSUS in 1994 [88].
Omission of ammonia results
Project MIX assessed ammonia levels in the smoke of the test cigarettes, yet the results were not included in the published report [16]. Internal reports from early 1999 [89],[90] show that ammonia was analyzed and significantly elevated in the smoke from ingredient group 1 high level and ingredient group 2 low and high levels, and significantly decreased in ingredient group 3 (containing menthol) low and high levels, compared to control cigarette smoke (Figure 1). The omission of ammonia test results from the final reports is curious in light of the fact that ammonia increases the pH of tobacco smoke making it less acidic and therefore easier to smoke [91] while increasing the bioavailability of the nicotine present in the smoke [92],[93]. No pH test results for the Project MIX test cigarettes were found.
![This result from an internal INBIFO report dated January 1999 shows results for measuring ammonia in Project MIX <em class="ref">[<b>89</b>]</em>.](https://www.prelekara.sk/media/cache/resolve/media_object_image_large/media/image/0387342876eceeaf287dc4875b44ce92.png)
Presentation of Results: Normalizing by Total Particulate Matter
In October 1998, “at the request of the client [PM USA]” [94], INBIFO scientists amended the Project MIX study protocol to report and analyze results on a per total particulate matter (TPM) basis; the original January 1998 plan [95] called for results to be presented on a per cigarette basis. The change was made 8 mo after INBIFO scientist Wolf Reininghaus reported to PM USA Manager of External Studies Gerry Nixon [96],[97] that the Project MIX “prototypes differ from control in a 10% higher TPM yield (which is compensated for by dilution in the subchronic study), [and] a 20% higher acrolein yield (which is, after compensation for TPM, no relevant difference)” [96].
The published paper does include a table reporting the smoke chemistry results on a per cigarette basis [16]; these data showed that all the test cigarettes containing any additives produced more TPM than the control cigarettes (ingredient group 1 low and high levels were 15% and 13% higher, ingredient group 2, 17% and 28%, and ingredient group 3, 13% and 16%, higher). Rather than emphasizing the biological importance of this increase in TPM, PM's researchers normalized the results by TPM and discussed whether the additives increased the amounts of each toxin in the smoke per unit TPM produced compared to the control cigarettes. Thus, as long as the amount of a toxin in the smoke of a test cigarette increased by less than the amount TPM increased in that cigarette, the ratio would drop even if both the toxin and TPM increased with the additives.
After TPM normalization, the graphical presentation of results as radar plots in the published paper [16] shows only five of the 31 toxins increased in ingredient group 1 and 15 showed drops. The PM scientists concluded, on the basis of this analysis (and similar analyses for the other two ingredient groups), “that the addition of these 333 commonly used ingredients to cigarettes in three groups did not add to the toxicity of smoke, even at the exaggerated levels tested in the present series of studies” [16].
Smokers do not, however, smoke cigarettes to titrate their TPM exposure. They smoke whole cigarettes, generally to obtain a certain amount of nicotine [98]–[100]. Therefore we used PM's published results [16] to prepare a corresponding set of radar plots that present the levels of toxins per cigarette (as a fraction of control) and the ratio of the levels of toxin per unit nicotine for the cigarettes with additives compared to the level of toxin per unit nicotine in the control cigarettes (Figures 2–4). Presenting the Project MIX results this way provides a much different picture than that in PM's paper: On a per cigarette basis, 31 of 51 chemicals increased in at least one of the three ingredient groups over control (with 17 decreased), and 37 increased (and nine decreased) on a per unit nicotine basis.
Fifteen chemicals increased by 20% or more above the levels observed in the control cigarettes (Table 1). These chemicals include a number of human and animal carcinogens (arsenic, cadmium, 1,3-butadiene, lead, formaldehyde, and PAHs), respiratory irritants (e.g., acrolein), and cellular toxicants (hydrogen cyanide, carbon monoxide). The number of smoke constituents that increase by 20% or more rises from seven constituents when normalized by TPM to 18 when expressed per cigarette and 23 on a per nicotine basis. Likewise, 24 chemicals fell by more than a 20% reduction when normalized by TPM (all but nine are on the published radar plots), compared to 12 on a per cigarette basis and nine on a per nicotine basis (Figures 2–4).
For unexplained reasons, the paper [16] excluded 19 of the 51 chemicals tested from the radar plots that formed the basis for discussing the results (1, Table S-2). Two of the excluded compounds significantly increased in smoke on a per cigarette basis of additive-added cigarettes, compared to smoke of control cigarettes, for all ingredient groups: benzo[-]fluoranthenes and benzo[ghi]perylene, both PAHs. Six more PAHs that were significantly higher in the smoke from cigarettes of ingredient groups 2 and 3 were also missing from the published radar plots—phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, and chrysene—as well as a seventh, indeno[1,2,3-cd]pyrene, which was significantly increased in ingredient group 1. (We include all 51 smoke constituents in our analysis, shown in Figures 2–4 and Table 1.).
In Vivo Subchronic Inhalation Testing
The in vivo animal toxicology portion of Project MIX consisted of exposing male and female Sprague-Dawley rats, nose only, to either fresh air (sham) or diluted mainstream smoke from the Project MIX control and test cigarettes for 90 d, followed by a 42-d postexposure period, after which time the animals were sacrificed and histopathology studies were completed [18]. The levels of smoke exposure were adjusted to expose all rats to the same level of TPM (150 µg TPM/l), regardless of which cigarettes (i.e., control or ingredient group) were used to generate the smoke.
The PM investigators [18] reported a “few minor differences” in the biological activity of the rats exposed to cigarettes with the additives, but dismissed these differences as “following no clear pattern,” suggesting they are “due to statistical inference.” The overall conclusion, reported in the paper's abstract is that “the data [from a 90-d inhalation study] indicate that the addition of the 333 commonly used ingredients, added to cigarettes in three groups, did not increase the inhalation toxicity of the smoke.”
The animal toxicology results [18] reported from Project MIX were based on a small number of rats in each experiment (usually nine), raising the possibility that the failure to detect statistically significant changes in the end points were due to underpowering the experiments rather than lack of a real effect. To explore this possibility, we assumed that the published descriptive statistics (means and standard deviations [SDs]) were unbiased estimates of the effects of those additives, then examined what the statistical conclusions would have been had Project MIX found these results on the basis of a sample size of 50 rats per group rather than nine rats per group. (While doing the actual experiment with a larger sample size would likely not yield precisely the same means and SDs as in the published paper, the differences should be randomly distributed on the assumption that the reported results are unbiased). We selected a sample size of 50 because PM used a much larger sample size of 99 rats (of each gender) in its study comparing the effects of secondhand smoke with diesel exhaust [101],[102]. When the reported standard error of the mean (SEM) was 0, we assumed the SD was 0.001. We then computed an analysis of variance using the reported means and SDs with an n of 50; if this ANOVA was significant (p<0.05), we then conducted multiple comparisons against control cigarettes with Holm-Sidak t-tests using a 0.05 family error rate. All calculations were done with Primer of Biostatistics (version 6) with the n, mean, SD option for ANOVA [103].
This exercise yielded 194 statistically significant changes (compared to the 26 reported in PM's published paper, Text S1,Table S-3), suggesting that a better powered study would have detected a much broader range of biological effects associated with the additives than identified in PM's published paper [18], suggesting that it substantially underestimates the toxic potential of cigarette smoke and additives.
Discussion
The laboratory aspects of Project MIX studies appear to have been conducted using well-accepted laboratory practices, so the raw data are probably unbiased. The essential conclusion that the PM investigators reached, on the basis of these data, that “The studies with ingredients added to cigarettes did not demonstrate any meaningful effect of the ingredients on the toxicity of cigarettes” [15], however, is a reflection of the way that the data were normalized in the studies of toxins in the smoke [16] and low power of the animal toxicology studies [18]. Despite these problems, Project MIX results have been widely promoted to the scientific community [104]–[116], the public at large, and the Institute of Medicine [117], as well as cited by other tobacco industry scientists [118]–[122].
The conclusion that the additives did not increase the yield of toxins in the smoke is a direct result of the fact that the cigarettes containing the additives produced 15%–28% more TPM than the control tobacco-only cigarettes. The fact that the additives lead to more TPM is, itself, an important indicator of increased toxicity because the TPM in cigarette smoke leads to substantial increases in risk of cardiovascular disease, with a steep and highly nonlinear dose-response at low levels of exposure [123]–[126]. TPM exposure also disrupts physiological angiogenesis and contributes to ectopic pregnancy, spontaneous abortion, preterm delivery, sudden infant death syndrome, and slower wound healing [127].
In a letter to the editor of Food and Chemical Toxicology, Vleeming et al. [128] also commented on the fact that normalizing toxin production in smoke gave a misleading picture of the effects of the additives on smoke toxicity. They suggested that toxin levels should be normalized by the amount of tobacco in each cigarette. Since PM reduced the amount of tobacco when including the additives to maintain the mass of the cigarette constant, presenting the results normalized by tobacco weight led to even greater increases in the estimated effects of the toxins than we present (in our Figures 2–4). The PM researchers justified their use of TPM normalization on the grounds that “We choose TPM as our basis of comparison to be consistent with the animal and in vitro studies presented in this series of publications” and because “Since consumers choose to smoke cigarettes according to the tar delivery and taste, we chose to normalize the data to the TPM yield to reveal the effect of ingredients which contribute to the taste” [129]. The first of these arguments is surprising since, as noted above, the original design of Project MIX did not anticipate reporting and analyzing results normalized by TPM [130] and only introduced this normalization after PM had results showing that the additives led to higher levels of TPM (even though there was correspondingly less tobacco in the cigarettes). Also, as noted above, it is well-established that smokers smoke to control the delivery of nicotine, not tar (TPM) [98]–[100] and normalizing toxin deliveries by nicotine delivery yields higher toxicity estimates than normalizing by TPM (Figures 2–4; Table 1).
The fact that the in vivo toxicology studies [18] were conducted at matched levels of TPM for the smoke from all cigarettes (to hold TPM constant) also meant, as PM scientist Reininghaus noted internally in 1998 [96], that the rats breathing the smoke from the cigarettes with the additives were exposed to lower levels of toxins in the smoke than if the exposures had been matched on another smoke variable, such as nicotine delivery. To the extent that the mass of tobacco in the cigarette determines nicotine delivery, Vleeming et al.'s [128] approach suggests that the underestimates of toxin exposures the rats received compared to human smokers could be substantial.
Another problem with the in vivo toxicology studies is that PM exposed the animals to fixed levels of TPM and the ratio of TPM to gas phase toxins changes with the different ingredient groups. If the production of gas phase toxins increases less than the increase in TPM, then exposing the rats to a fixed amount of TPM will reduce exposure to the gaseous constituents. For physiological endpoints affected by these gases the toxicity might appear to decrease on the basis of per TPM versus per cigarette exposures. Indeed, when PM instructed INBIFO to analyze the smoke chemistry data on a per TPM basis [94] they noted that such an adjustment was already “compensated for by dilution in the subchronic study” [96].
The way the animal toxicology studies were designed with the relatively short (90 d) exposure period and follow-up after the end of the exposure (42 d) also raises concerns. PM's comparison of secondhand smoke with diesel exhaust (also conducted at INBIFO) had 99 rats of each gender in each exposure group, exposed the rats for 24 mo (730 d) with a 6-mo (183 d) follow-up [101],[102]. Longer exposure and follow-up would have increased the power of Project MIX to detect toxic effects of the additives. Even with these downward biases due to experimental design, however, our results suggest that an adequately powered design would have revealed a large number of toxic effects on the rats.
PM conducted extensive research on individual additives; the industry documents contain a list of 170 projects [131] conducted between 1973 and 1995 that includes many single additive projects. These single additive projects include INBIFO Project Juice, in which citric acid (one of the 333 ingredients in the present study) was added in low (2.6%), medium (3.9%), and high (6.3%) quantities to cigarette filler [132]. The amounts of citric acid reported for Project MIX ingredient group 1 were low level = 44 ppm, and high level = 122 ppm. It is not clear how these amounts of citric acid compare across studies. Studies were also conducted with lactic acid (Project Milk [133]), propylene glycol [134], and cocoa [34]. These studies were also conducted using low ratios between groups, along the order of only a 2- to 3-fold difference.
Project MIX also included in vitro genotoxicity and cytotoxicity studies [17] using a standard test panel PM developed in 1996, including the Ames test [14],[135]–[137]. Not surprisingly, the results of these tests found that all of the cigarettes, whether or not they included additives, were genotoxic and mutagenic. These tests, however, are screening tests, not sensitive measures of dose-response [138]–[141]. Therefore they are not appropriate for quantifying changes in toxicity associated with the additives. Absent the unlikely situation that the additives would eliminate tobacco smoke's genotoxicity and mutagenicity, the failure to find increased toxicity associated with the additives does not support the conclusion that “in vitro mutagenicity and cytotoxicity of the cigarette smoke were not increased by the addition of the ingredients” [17].
Scientists at British American Tobacco (BAT) conducted a similar project that in 2004 was also published in Food and Chemical Toxicology [142]–[144]. The BAT studies assessed 482 additives in various mixtures “at or above their typical maximum levels used on cigarettes sold by BAT” through an analysis of 45 selected smoke constituents in mainstream smoke of test cigarettes; in vitro mutagenicity and cytotoxicity tests (Ames test, mammalian cell micronucleus test, and neutral red uptake test) and 90-d inhalation studies in male and female Sprague-Dawley rats (ten rats per group). Using comparable rates of exposure of rats to smoke (approximately 20% less than the PM study rats), the BAT scientists report similar deleterious histopathological findings as Project MIX: increased density of goblet cells in respiratory epithelium, increased hyperplasia/degeneration of the nasal cavity, and increased epiglottal metaplasia. Similar to the PM scientists, the BAT scientists downplayed the findings by concluding that they were inconsistent, indicating that the response of rats exposed to smoke was indistinguishable between test and control cigarettes.
The kind of manipulation of the presentation of scientific results demonstrated by the publication of the Project MIX results [15]–[18], is nothing new for the tobacco industry; industry researchers have a long history of doing so around a variety of issues related to secondhand smoke [145]–[149]. While the procedures to collect the data themselves appear sound, the way that the data were analyzed and interpreted is not. An important implication of the analysis we present is that the scientific community and regulatory authorities cannot take the conclusions in tobacco industry (or industry-funded) research or research published in industry-dominated journals such as Food and Chemical Toxicology at face value. Vigorous implementation of FCTC article 5.3, which seeks to protect the policy making process against tobacco industry interference and manipulation [150], underscores the need for such skepticism in considering research such as Project MIX (and the corresponding papers from BAT) at face value in the rulemaking process. It will be important for the US FDA, WHO, and regulatory agencies in other countries who are working to implement FCTC articles 9–11 to insist on receiving all drafts of the study protocol (taking particular care to not allow the tobacco companies to use lawyer involvement in the process as a way to avoid disclosure) together with the raw data to reduce the likelihood of the problems identified in this paper.
If one accepts PM's assertion that Project MIX evaluated additives in groups that “resemble typical commercial blended cigarettes” [15], the data PM collected could be used for policy making regarding the use of these 333 additives. That the Project MIX scientists examined cigarette additives in combination (rather than singly) allows for the possibility that the additives act either synergistically or antagonistically. Allowing for this synergism is important. A comparison of the estimated lung cancer effect of tobacco smoke produced by summing the individual effects of constituents of tobacco smoke produced an estimate of cancer risk much smaller than the observed epidemiological risk [114]. Probably this is because all the carcinogens and cardiac toxins in tobacco smoke have not been identified and because there are likely interactions between the different constituents in the complex mixture that cigarette smoke represents.
It is also important to emphasize that the definition of “harm” implicit in PM's Project MIX and BAT's similar experiments [142]–[144]—direct short-term toxic effects of additives—is just one dimension of the effects that additives have on disease. Additives also have an effect on product palatability and pharmacologic properties (Box 1) in ways that make cigarettes less harsh and more pleasant to the user [3],[151],[152], leading to higher initiation and lower cessation and, so, more tobacco use and tobacco-induced disease [4],[19]. Indeed, it was primarily on the basis of these population-level impacts that the FDA Tobacco Products Scientific Advisory Committee concluded that, “Removal of menthol cigarettes from the marketplace would benefit public health in the United States” [19].
Limitations
As noted in the Methods, this study is based on the documents that PM made available as a result of litigation against the company. While we were able to find several drafts and detailed results of the Project MIX studies, that, for example, allowed us to determine that the protocol for analyzing the data was altered after the results indicating increased TPM were obtained, there are key missing pieces of information: why PM chose the additives that they did for study, as well as the levels and combinations and the reason for selecting the sample sizes in the toxicology studies. This situation represents a limitation of the available sources of information and highlights the importance of the FDA using its new authority to obtain all sources of data on additive testing and other features of product manufacturing and testing and to make them fully available for review.
Conclusion
The analysis in this paper shows that many of the toxins in cigarette smoke increase substantially when additives are put in cigarettes, including the level of TPM, and that, assuming that the toxicological results from Project MIX represent unbiased estimates of the true biological effects, these data show many adverse biological consequences (and that the failure to reach statistical significance was the result of underpowered studies rather than lack of an effect). In particular, regulatory authorities, including the FDA and similar agencies elsewhere who are implementing FCTC articles 9–11, could use the Project MIX data to eliminate the use of these 333 additives (including menthol, which is the major component of ingredient group 3) in cigarettes.
Any tobacco company would, of course, remain free to submit an application to the FDA, or other regulatory agency, to reintroduce use of an additive if they could provide convincing data from adequately powered studies that the additive truly did not have any adverse health consequences.
Supporting Information
Zdroje
1. WakehamH Hazleton Laboratories. 22 Jul 1963. Bates number 1001606422. Available: http://legacy.library.ucsf.edu/tid/kps08e00. Accessed 14 November 2011
2. Cigarette Labeling and Advertising Act (P.L. 98-474). Available: http://uscode.house.gov/download/pls/15C36.txt. Accessed 14 November 2011
3. World Health Organization Partial guidelines for implementation of Articles 9 and 10 of the WHO Framework Convention on Tobacco Control (Regulation of the contents of tobacco products and Regulation of tobacco product disclosures). Available: http://www.who.int/fctc/guidelines/Decisions9and10.pdf. Accessed 4 June 2011
4. LeeYOGlantzSA 2011 Menthol: putting the pieces together. Tob Control 20 ii1 ii7
5. 2009 Family Smoking Prevention and Tobacco Control and Federal Retirement Reform. US Public Law 111-31. Available: http://www.gpo.gov/fdsys/pkg/PLAW-111publ31/pdf/PLAW-111publ31.pdf. 111th Congress. Washington (D.C.): G.P.O. Accessed 14 November 2011
6. McDanielPAMaloneRE 2005 Understanding Philip Morris's pursuit of US government regulation of tobacco. Tob Control 14 193 200
7. McDanielPASmithEAMaloneRE 2006 Philip Morris's Project Sunrise: weakening tobacco control by working with it. Tob Control 15 215 223
8. GlantzSABarnesREubanksSY 2009 Compromise or capitulation? US Food and Drug Administration jurisdiction over tobacco products. PLoS Med 6 e1000118 doi:10.1371/journal.pmed.1000118
9. US Food and Drug Administration, Department of Health and Human Services 2009 Candy and fruit flavored cigarettes now illegal in United States; step is first under new tobacco law. Available: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm183211.htm. Accessed 24 July 2011
10. ReesVWKreslakeJMO'ConnorRJCummingsKMParascandolaM 2009 Methods used in internal industry clinical trials to assess tobacco risk reduction. Cancer Epidemiol Biomarkers Prev 18 3196 3208
11. World Health Organization WHO Framework Convention on Tobacco Control. Available: http://www.who.int/fctc/en/index.html. Accessed 12 September 2008
12. EllisCLMilbyDL 1997 970000 FDA Compliance Preparation. 9 Jul 1997. Bates number 2078468223/2078468224. Available: http://legacy.library.ucsf.edu/tid/asl75c00. Acccessed14 November 2011
13. DiethelmPARielleJCMcKeeM 2005 The whole truth and nothing but the truth? The research that Philip Morris did not want you to see. Lancet 366 86 92
14. NixonG 21 Aug 1997. Bates number 2501950681/2501950682. Available: http://legacy.library.ucsf.edu/tid/gke81c00. Accessed 14 November 2011
15. CarminesEL 2002 Evaluation of the potential effects of ingredients added to cigarettes. Part 1: cigarette design, testing approach, and review of results. Food Chem Toxicol 40 77 91
16. RustemeierKStabbertRHaussmannHJRoemerECarminesEL 2002 Evaluation of the potential effects of ingredients added to cigarettes. Part 2: chemical composition of mainstream smoke. Food Chem Toxicol 40 93 104
17. RoemerETewesFJMeisgenTJVeltelDJCarminesEL 2002 Evaluation of the potential effects of ingredients added to cigarettes. Part 3: in vitro genotoxicity and cytotoxicity. Food Chem Toxicol 40 105 111
18. VanscheeuwijckPMTeredesaiATerpstraPMVerbeeckJKuhlP 2002 Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity. Food Chem Toxicol 40 113 131
19. FDA Tobacco Products Scientific Advisory Committee Menthol cigarettes and public health: review of the scientific evidence and recommendations (as reviewed at the TPSAC meeting on March 18, 2011). Available: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/TobaccoProductsScientificAdvisoryCommittee/UCM247689.pdf. Accessed 17 April 2011
20. Product Integrity, Philip Morris USA 2001 Evaluation of menthol for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067617005/2067617095. Available: http://legacy.library.ucsf.edu/tid/nox75a00. Accessed 14 November 2011
21. Product Integrity, Philip Morris USA 2001 Evaluation of cocoa for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067637337/2067637404. Available: http://legacy.library.ucsf.edu/tid/wcc34a00. Accessed 14 November 2011
22. Philip Morris USA 2001 Evaluation of propylene glycol for use as a cigarette ingredient. 03 Oct 2001. Bates number 2085543332/2085543389. Available: http://legacy.library.ucsf.edu/tid/rrn43a00. Accessed 14 November 2011
23. Product Integrity, Philip Morris USA 2001 Evaluation of vanilla extract for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067617286/2067617380. Available: http://legacy.library.ucsf.edu/tid/lox75a00. Accessed 14 November 2011
24. Product Integrity, Philip Morris USA 2001 Evaluation of glycerol for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067636691/2067636778. Available: http://legacy.library.ucsf.edu/tid/jec34a00. Accessed 14 November 2011
25. Product Integrity, Philip Morris USA 2001 Evaluation of sweet orange oil for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067617122/2067617209. Available: http://legacy.library.ucsf.edu/tid/mox75a00. Accessed 14 November 2011
26. Product Integrity, Philip Morris USA 2001 Evaluation of licorice extract for use as a cigarette ingredient. 03 Oct 2001. Bates number 2067661381/2067661422. Available: http://legacy.library.ucsf.edu/tid/rha34a00. Accessed 14 November 2011
27. US Department of Health & Human Services 2011 Tobacco Products Scientific Advisory Committee. US Food and Drug Administration. Available: http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/TobaccoProductsScientificAdvisoryCommittee/default.htm. Accessed 10 December 2010
28. HeckJD 2010 A review and assessment of menthol employed as a cigarette flavoring ingredient. Food Chem Toxicol 48 S1 38
29. AndersonSJMcCandlessPMKlausnerKTaketaRYergerVB 2011 Tobacco documents research methodology. Tobacco Control 20 ii8 ii11
30. BeroL 2003 Implications of the tobacco industry documents for public health and policy. Annu Rev Public Health 24 267 288
31. MaloneREBalbachED 2000 Tobacco industry documents: treasure trove or quagmire? Tob Control 9 334 338
32. GlantzSABarnesDEBeroLHanauerPSladeJ 1996 The cigarette papers Berkeley University of California Press 219 224
33. Author unknown [Document from Thomas Osdene's files at Philip Morris] 1978 Test Protocol LTF. 13 Feb 1978. Bates number 1000024814/1000024816. Available: http://legacy.library.ucsf.edu/tid/cge97e00. Accessed 14 November 2011
34. KuhnDRomerESchmatzETeredesaiAThomasC 1982 Report a 0500/3023. Comparative study on whole smoke condensates from cigarettes with and without addition of cocoa in the mouse skin painting assay. 01 Mar 1982. Bates number 2028790638/2028790972. Available: http://legacy.library.ucsf.edu/tid/zkz74e00. Accessed 14 November 2011
35. BlakeCBoschVDrozAPossetL 1987 The effect of the humectants glycerol and propylene glycol on mainstream and sidestream smoke deliveries of acrolein, formaldehyde, acetaldehyde, acetone and propionaldehyde. 00 Feb 1987. Bates number 2023186677/2023186680. Accessed 14 November 2011
36. GenoudYLecoultreEMoserFMurrayMPiadeJJ 1982 Project title: Analytical Investigations 820417–820518. 27 May 1982. Bates number 2501308073/2501308074. Available: http://legacy.library.ucsf.edu/tid/wyc29e00. Accessed 14 November 2011
37. BlakeCJLoseeBOeyJPossetLRychenerM 1986 Final report project slow 77-c1 chemical and biological testing of prototype cigarettes 72-c1 and 77-C1. 10 Jan 1986. Bates number 2022150326/2022150352. Available: http://legacy.library.ucsf.edu/tid/yyd59e00. Accessed 14 November 2011
38. StinnW 1989 Concept. 23 May 1989. Bates number 2028991555/2028991564. Available: http://legacy.library.ucsf.edu/tid/kmf69e00. Accessed 14 November 2011
39. GugelHHeilMOeyJRomerEZzdroe 1982 Proposal a 0268/2074 4-day cytotoxicity study exposure to whole smoke and gas/vapor phase of cigarettes coral-a and coral-b, and of standard reference cigarette 2r1f of human lung cells pan-L3. 00 1982. Bates number 2501217555/2501217604. Available: http://legacy.library.ucsf.edu/tid/zju32e00. Accessed 14 November 2011
40. ReininghausW 1990 Achievements and proposed research projects. 15 Nov 1990. Bates number 2029173277/2029173308. Available: http://legacy.library.ucsf.edu/tid/ndx37e00. Accessed 14 November 2011
41. MorrisPhilip 1984 Table sidestream smoke doses in in vivo inhalation studies. 10 Jul 1984. Bates number 2501668618. Available: http://legacy.library.ucsf.edu/tid/vin49e00. Accessed 14 November 2011
42. BachmayerD 1989 Concept sister chromatid exchange and chromosome aberration inducing activities of ethylnitrosourea and formaldehyde in nasal and tracheal epithelial cells: acute sub acute study on rats (Pt). 00 1989. Bates number 2028826323/2028826333. Available: http://legacy.library.ucsf.edu/tid/kco27e00. Accessed 14 November 2011
43. DoullJFrawleyJPGeorgeWLoomisTSquireRA 1994 A safety assessment of ingredients added to tobacco in the manufacture of cigarettes. 00 Mar 1994. Bates number 2073910155/2073910163. Available: http://legacy.library.ucsf.edu/tid/acm05a00. Accessed 14 November 2011
44. American Tobacco Company, Brown & Williamson Tobacco Corporation, Liggett Group Inc., Lorillard Inc., Philip Morris Incorporated et al. 1993 List of ingredients added to tobacco in the manufacture of cigarettes (as of 931231). 31 Dec 1993. Bates number 89284138/89284169. Available: http://legacy.library.ucsf.edu/tid/ayy10e00. Accessed 14 November 2011
45. Tobacco Reporter 1994 Flavorings. Safe. cig flavorings not a risk, says independent panel. Tr Staff Report. Jul 1994. Bates number 523319071/523319078. Available: http://legacy.library.ucsf.edu/tid/sis20d00. Accessed 14 November 2011
46. MorrisPhilip 1998 6 June 1998. Bates number 2501826284/2501826291. Available: http://legacy.library.ucsf.edu/tid/sac90c00. Accessed 14 November 2011
47. CarminesEL 2001 L.S.R.O. Presentation. 06 Dec 2001. Bates number 2067636959/2067637071. Available: http://legacy.library.ucsf.edu/tid/ekw86a00. Accessed 14 November 2011
48. Philip Morris Incorporated 2011 Philip Morris glossary of names. http://legacy.library.ucsf.edu/glossaries/pm_gloss_bo.jsp. Accessed 16 April 2011
49. Philip Morris Incorporated 1999 Letter to Borzelleca confirming agreement to serve on PM's Scientific Advisory Board. 02 Nov 1999. Bates number 3001109471. Available: http://legacy.library.ucsf.edu/tid/csu91g00. Accessed 14 November 2011
50. Philip Morris Incorporated 1995 Letter to Houghton Sr. (Consultant agreement for Borzelleca). 13 Jan 1995. Bates number 2059674110. Available: http://legacy.library.ucsf.edu/tid/hds22d00. Accessed 14 November 2011
51. BorzellecaJCoxRH Amendment number 1 to agreement dated 19981001 between Pharmacolo & Toxicology, Inc. And Philip Morris Incorporated. 08 Jun 1999. Bates number 3001337718–3001337719. Available: http://legacy.library.ucsf.edu/tid/lem71h00. Accessed 14 November 2011
52. 1991 International conference: priorities for indoor air research and action. Bates number 400083804–400083813. Available: http://legacy.library.ucsf.edu/tid/wwq52a99. Accessed 14 November 2011
53. GarneDWatsonMChapmanSByrneF 2005 Environmental tobacco smoke research published in the journal Indoor and Built Environment and associations with the tobacco industry. Lancet 365 804 809
54. TongEKEnglandLGlantzSA 2005 Changing conclusions on secondhand smoke in a sudden infant death syndrome review funded by the tobacco industry. Pediatrics 115 e356 366
55. RJRColucciAV 1985 BYU ETS Project. 16 May 1985. Bates number 507915853. Available: http://legacy.library.ucsf.edu/tid/tig14d00. Accessed 14 November 2011
56. R.J. Reynolds Tobacco Company 2000 R&D Scientific Presentations. 11 Aug 2000. Bates number 531630656/531630753. Available: http://legacy.library.ucsf.edu/tid/knp55a00. Accessed 14 November 2011
57. LoweJEMooreKE 1987 Molecular biology of tumor promotion. Approved Ar #87-656. Internal # Fs 7067. 23 Dec 1987. Bates number 506857456. Available: http://legacy.library.ucsf.edu/tid/twf55a00. Accessed 14 November 2011
58. DenobleVJDraganYPKornfeldRMelePCNaworalJ 1986 Chronic nicotine administration. Sep 1986. Bates number 2047340356/2047340376. Available: http://legacy.library.ucsf.edu/tid/gyc36e00. Accessed 14 November 2011
59. Philip Morris USA Research Center 1983 1610 - Behavioral Pharmacology Annual Report 830000. 00 1983. Bates number 1003178534/1003178615. Available: http://legacy.library.ucsf.edu/tid/gpd74e00. Accessed 14 November 2011
60. MorrisPhilip 1999 Publications from RD and E. 21 Apr 1999. Bates number 2079072311/2079072423. Available: http://legacy.library.ucsf.edu/tid/bjd47c00. Accessed 14 November 2011
61. AllisonJ 2001 Fw: SAB Members. 09 Feb 2001. Bates number 2085523903. Available: http://legacy.library.ucsf.edu/tid/tcs10c00. Accessed 14 November 2011
62. BarbaraR 2007 Colloquia_1979-2007.pdf. 28 Feb 2007. Bates number 3063033715–3063033749. Available: http://legacy.library.ucsf.edu/tid/hqy91g00. Accessed 14 November 2011
63. SmithG 1987 TAC Visit to Dr Barry Halliwoll University of London King's College 26th November 1987. 02 Dec 1987. Bates number 400959753–400959754. Available: http://legacy.library.ucsf.edu/tid/bge04a99. Accessed 14 November 2011
64. FisherA-L 2000 Risk Assessment Forum - First Meeting. 03 Nov 2000. Bates number 325381946. Available: http://legacy.library.ucsf.edu/tid/sry61a99. Accessed 14 November 2011
65. FisherA-L 2000 Risk Assessment Meeting - 30th November. 08 Nov 2000. Bates number 325381990–325381991. Available: http://legacy.library.ucsf.edu/tid/ssy61a99. Accessed 14 November 2011
66. Council for Tobacco Research 1978 Microbiological Associates Contract 14-D July 1, 1977- June 30, 1978 - “the in vivo and in vitro analyses of the biological effects of smoke-related chemicals.” 30 Jun 1978. Bates number 10007057–10007058. Available: http://legacy.library.ucsf.edu/tid/vjd66b00. Accessed 14 November 2011
67. HenryCKouriR 1982 Associates M. Letter to Sheldon Sommers (Council for Tobacco Research). 27 Jan 1982. Bates number SF0828041/0828045. Available: http://legacy.library.ucsf.edu/tid/pfj10a00. Accessed 14 November 2011
68. Council for Tobacco Research 1980 Publication by Grantee; interindividual variations of epoxide hydratase activity in human liver and lung biopsies, lymphocytes and fibroblast cultures; microsomes, drug oxidations, and chemical carcinogenesis. Vol. Ii. 18 Aug 1980. Bates number HK1361092/1361093. Available: http://legacy.library.ucsf.edu/tid/abm2aa00. Accessed 14 November 2011
69. MuschollE (Univ Mainz)OeschF (Univ Mainz) 1978 Application for renewal of research grant importance of epoxide hydratase, dihydrodiol dehydrogenase and conjugating enzymes in the control of mutagenic and transforming metabolites of polycyclic hydrocarbons occurring in cigarette smoke and interindividual comparison of their activities in man. 28 Jun 1978. Bates number 50124698/50124707. Available: http://legacy.library.ucsf.edu/tid/sdh36d00. Accessed 14 November 2011
70. WalkRA 1994 Visit by Dr. Mathuros Ruchirawat, Vice President for Research, Chulabhorn Research Institute (Cri), Bangkok, Thailand. 13 Jul 1994. Bates number 2062045574/2062045576. Available: http://legacy.library.ucsf.edu/tid/vzz43e00. Accessed 14 November 2011
71. SaundersSSteeleHApoptosis SubcommC 1999 Apoptosis Working Group Meeting Report. 18 Mar 1999. Bates number 521559705/521559706. Available: http://legacy.library.ucsf.edu/tid/vrp60d00. Accessed 14 November 2011
72. SmithG 1990 International Symposium on Nicotine. Hamburg June 1990. 09 Jul 1990. Bates number 300522856–300522865. Available: http://legacy.library.ucsf.edu/tid/zks18a99. Accessed 14 November 2011
73. WalkRA 4 Jul 1991 Bates number 2028698861. Available: http://legacy.library.ucsf.edu/tid/yck12e00. Accessed 14 November 2011
74. ThorntonR 1993 Research proposal. 10 Sep 1993. Bates number 401015140. Available: http://legacy.library.ucsf.edu/tid/kgr61a99. Accessed 14 November 2011
75. Scientific Research Group 1998 Projects expenditure. 00 1998. Bates number 321727559. Available: http://legacy.library.ucsf.edu/tid/qrr44a99. Accessed 14 November 2011
76. RudgeL 1994 Note from L Rudge to A Sarkar regarding the possibility of ITC sponsoring scientists to attend the International Cancer Congress. 06 May 1994. Bates number 500863892–500863894. Available: http://legacy.library.ucsf.edu/tid/qdh10a99. Accessed 14 November 2011
77. BakerR 1999 British American Tobacco Scientific Research Group. 19 Aug 1999. Bates number 325284711–325284720. Available: http://legacy.library.ucsf.edu/tid/rcm82a99. Accessed 14 November 2011
78. CarminesEL 1999 Evaluation of the potential effects of flavor ingredients added to cigarettes. Part 1. Cigarette design and testing. 10 Apr 1999. Bates number 2505330385/2505330415. Available: http://legacy.library.ucsf.edu/tid/mke25c00. Accessed 14 November 2011
79. WinstonN 1998 Product testing laboratory final laboratory averages report none - 97-1018 - extramural flavor study, activity number t00481 large scale. 19 Jan 1998. Bates number 2075175965/2075175968. Available: http://legacy.library.ucsf.edu/tid/iey56c00. Accessed 14 November 2011
80. NixonG 1998 Fax Project Mix. 29 Jan 1998. Bates number 2075175964. Available: http://legacy.library.ucsf.edu/tid/hey56c00. Accessed 14 November 2011
81. LeeBCBurnsDMGairolaCGHarrisJEHoffmanD 1993 Toxicity testing plan: US Consumer Product Safety Commission in Consultation with the US Department of Health and Human Services Washington (D.C.) US Department of Health and Human Services
82. International Agency for Research on Cancer 1998 Tobacco smoking Lyon, France International Agency for Research on Cancer
83. SolanaR 1997 Chemistry list for product integrity testing. 18 Mar 1997. Bates number 2501778545. Available: http://legacy.library.ucsf.edu/tid/fpt93g00. Accessed 14 November 2011
84. 1993 Draft smoke chemistry testing list based on Hoffman list, IARC and Sg lists, and Advisory Board recommendations. Bates number 2501778546. Available: http://legacy.library.ucsf.edu/tid/rqp93g00. Accessed 14 November 2011
85. Agency for Toxic Substances & Disease Registry Centers for Disease Control. Toxicity of polycyclic aromatic hydrocarbons. Available: http://www.atsdr.cdc.gov/csem/pah/pah_physiologic-effects.html. Accessed November 23, 2010. [An archived copy of this page is available on the Internet Archive at http://web.archive.org/web/20110606032914/http://www.atsdr.cdc.gov/csem/pah/pah_physiologic-effects.html] Accessed 14 November 2011
86. California Office of Environmental Health Hazard Assessment 2001 Polycyclic organic matter. http://oehha.ca.gov/air/toxic_contaminants/pdf_zip/PAHs_Final.pdf. Accessed 14 November 2011
87. PelkonenONebertDW 1982 Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol Rev 34 189 222
88. MorrisPhilip 1994 [Information on the standard design of our 21-day inhalation studies with cigarette smoke and on results obtained for URSUS-35]. 00 Sep 1994. Bates number 2501383813/2501383841. Available: http://legacy.library.ucsf.edu/tid/uhk42d00. Accessed 14 November 2011
89. INBIFO/CRC 1999 Project Mix - chemical analysis of mainstream smoke. January 1999. Bates number 2505383586/2505383604. Available: http://legacy.library.ucsf.edu/tid/yfe39c00. Accessed 14 November 2011
90. GommWReininghausWRustemeierKVonholtK 1999 Report P 0500/3283. 04 Mar 1999. Bates number 2075165366/2075165429. Available: http://legacy.library.ucsf.edu/tid/tcb27d00. Accessed 14 November 2011
91. ReynoldsRJ 2009 Ammoniation. Bates number 509018864–509018865A. Available: http://legacy.library.ucsf.edu/tid/xxb66b00. Accessed 14 November 2011
92. StevensonTProctorR 2008 The secret and soul of Marlboro: Phillip Morris and the origins, spread, and denial of nicotine freebasing. Am J Public Health 98 1184 1194
93. BlevinsRAJr 1973 U.S Exhibit 20,669, Memo, re: Free nicotine as a difference in performance between brands, RAYMOND A. BLEVINS, JR., R.J. REYNOLDS TOBACCO CO., July 12, 1973. 12 Jul 1973. Bates number USX006703. Available: http://legacy.library.ucsf.edu/tid/acq36b00. Accessed 14 November 2011
94. RustemeierK 1998 Study plan amendment: P - 3283 Change-no.: 2 chemical analysis, mix. 09 Oct 1998. Bates number 2505104535/2505104536. Available: http://legacy.library.ucsf.edu/tid/pvw46c00. Accessed 14 November 2011
95. GommWHackenbergUReininghausWRustemeierKVonckenP 1998 Study plan P 0500/3283 chemical analysis of mainstream smoke of cigarettes containing ingredient mixtures. 29 Jan 1998. Bates number 2501958943/2501958963. Available: http://legacy.library.ucsf.edu/tid/cnf81c00. Accessed 14 November 2011
96. ReininghausW 1998 Telefax message no. 2248. 12 Feb 1998. Bates number 2075175916. Available: http://legacy.library.ucsf.edu/tid/dey56c00. Accessed 14 November 2011
97. ReininghausW 1998 Report P - 3283 table mainstream smoke yields of research cigarettes. 12 Feb 1998. Bates number 2505104541/2505104544. Available: http://legacy.library.ucsf.edu/tid/uvw46c00. Accessed 14 November 2011
98. USDHHS 1988 The health consequences of smoking: nicotine addiction. A report of the Surgeon General Washington (D.C.) US Department of Health and Human Services, Public Health Service, Centers for Disease Control, Center for Health Promotion and Education, Office on Smoking and Health DHHS Publication number (CDC) 88-8406, 1988 DHHS Publication number (CDC) 88-8406, 1988
99. National Cancer Institute 2001 Tobacco control monograph 13: risks associated with smoking cigarettes with low machine-measured yeilds of tar and nicotine Bethesda (Maryland) National Cancer Institute
100. BenowitzN 2010 Nicotine addiction. N Engl J Med 362 2295 2303
101. GommWHackenbergUKaeglerMKindtRKuhnD 1996 Study plan P 0500/3248 correlation of classic and mechanistic end points in a comparative tumorigenicity study on rats chronic effects of room-aged sidestream smoke and diesel engine exhaust (Pt). 09 Dec 1996. Bates number 2064276608/2064276713. Available: http://legacy.library.ucsf.edu/tid/yjo45d00. Accessed 14 November 2011
102. StinnWTeredesaiAAnskeitERustemeierKSchepersG 2005 Chronic nose-only inhalation study in rats, comparing room-aged sidestream cigarette smoke and diesel engine exhaust. Inhal Toxicol 17 549 576
103. GlantzSA 2005 Primer of biostatistics San Francisco McGraw-Hill
104. RoemerERustemeierKVanscheeuwijckPMMeisgenTJVeltelDJ 2000 Effects of the addition of flavor ingredients to the tobacco on the chemical composition and biological activity of cigarette smoke. 00 Mar 2000. Bates number 98984120. Available: http://legacy.library.ucsf.edu/tid/xsy64d00. Accessed 14 November 2011
105. ByrdDMIII 2004 (2004) Phase one: the feasibility of testing ingredients added to cigarettes Bethesda (Maryland) Life Sciences Research Office
106. ByrdDMIIILewisKDNixonPMFeldmanRS 2004 (2004) Phase two: scientific criteria for the evaluation of ingredients added to cigarettes Bethesda (Maryland) Life Sciences Research Office, Inc.
107. van der ToornMRezayatDKauffmanHFBakkerSJGansRO 2009 Lipid-soluble components in cigarette smoke induce mitochondrial production of reactive oxygen species in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 297 L109 L114
108. SobusJRMcCleanMDHerrickRFWaidyanathaSOnyemauwaF 2009 Investigation of PAH biomarkers in the urine of workers exposed to hot asphalt. Ann Occup Hyg 53 551 560
109. SobusJRMcCleanMDHerrickRFWaidyanathaSNylander-FrenchLA 2009 Comparing urinary biomarkers of airborne and dermal exposure to polycyclic aromatic compounds in asphalt-exposed workers. Ann Occup Hyg 53 561 571
110. SlebosDJRyterSWvan der ToornMLiuFGuoF 2007 Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death. Am J Respir Cell Mol Biol 36 409 417
111. SerdarBEgeghyPPWaidyanathaSGibsonRRappaportSM 2003 Urinary biomarkers of exposure to jet fuel (JP-8). Environ Health Perspect 111 1760 1764
112. MuttiACorradiMGoldoniMVettoriMVBernardA 2006 Exhaled metallic elements and serum pneumoproteins in asymptomatic smokers and patients with COPD or asthma. Chest 129 1288 1297
113. KimDAndersenMEChaoYCEgeghyPPRappaportSM 2007 PBTK modeling demonstrates contribution of dermal and inhalation exposure components to end-exhaled breath concentrations of naphthalene. Environ Health Perspect 115 894 901
114. FowlesJDybingE 2003 Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob Control 12 424 430
115. Al RashidiMShihadehASalibaNA 2008 Volatile aldehydes in the mainstream smoke of the narghile waterpipe. Food Chem Toxicol 46 3546 3549
116. MoennikesOVanscheeuwijckPMFriedrichsBAnskeitEPatskanGJ 2008 Reduced toxicological activity of cigarette smoke by the addition of ammonia magnesium phosphate to the paper of an electrically heated cigarette: subchronic inhalation toxicology. Inhal Toxicol 20 647 663
117. SolanaRPStrattonK 2000 Comment to IOM Question 3. 17 Apr 2000. Bates number 2082337716/2082337726. Available: http://legacy.library.ucsf.edu/tid/kim49c00. Accessed 14 November 2011
118. LemusRCarminesELVan MiertECogginsCRAnskeitE 2007 Toxicological comparisons of cigarettes containing different amounts of vanillin. Inhal Toxicol 19 683 699
119. GaworskiCLLemus-OlaldeRCarminesEL 2008 Toxicological evaluation of potassium sorbate added to cigarette tobacco. Food Chem Toxicol 46 339 351
120. GaworskiCLCogginsCRCarminesEL 2008 Palladium alters cigarette smoke toxicological profile, but accumulates in the lungs of rats during inhalation exposure. Inhal Toxicol 20 167 182
121. CarminesELLemusRGaworskiCL 2005 Toxicologic evaluation of licorice extract as a cigarette ingredient. Food Chem Toxicol 43 1303 1322
122. CarminesELGaworskiCL 2005 Toxicological evaluation of glycerin as a cigarette ingredient. Food Chem Toxicol 43 1521 1539
123. Institute of Medicine 2009 Secondhand smoke exposure and cardiovascular effects: making sense of the evidence Washington (D.C.) National Academy Press http://www.iom.edu/Reports/2009/Secondhand-Smoke-Exposure-and-Cardiovascular-Effects-Making-Sense-of-the-Evidence.aspx
124. PopeCA3rdBurnettRTKrewskiDJerrettMShiY 2009 Cardiovascular mortality and exposure to airborne fine particulate matter and cigarette smoke: shape of the exposure-response relationship. Circulation 120 941 948
125. US Department of Health and Human Services 2010 How tobacco smoke causes disease: the biology and behavioral basis for smoking-attributable disease. A report of the Surgeon General Washington (D.C.) Dept. of Health and Human Services, Public Health Service, Office of Surgeon General, 2010 Available: http://surgeongeneral.gov/library/tobaccosmoke/index.html
126. BrookRDRajagopalanSPopeCA3rdBrookJRBhatnagarA 2010 Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 121 2331 2378
127. EjazSInsan-ud-dinAshrafMNawazMLimC 2009 Cigarette smoke condensate and total particulate matter severely disrupts physiological angiogenesis. Food Chem Toxicol 47 601 614
128. VleemingWSchenkEOpperhuizenA 2004 Letter to the Editor. Food and Chemical Toxicology 42 865 866
129. CarminesELHaussmannH-JRustemeierKRoemerEStabberR 2004 Response to Letter. Food and Chemical Toxicology 42 867
130. GommWHackenbergUReininghausWRustemeierKVonckenP 1998 Study plan P 0500/3283 chemical analysis of mainstream smoke of cigarettes 97.Mg.292, 97.Mg.295, 97.Mg.296, and 97.Mj.125 to 97.Mj.128 Project Mix (Pt). 03 Feb 1998. Bates number 2502334950/2502334971. Available: http://legacy.library.ucsf.edu/tid/egh81c00. Accessed 14 November 2011
131. INBIFO 1998 INBIFO testing index - by project number 23 Feb 1998. Bates number 2075175385/2075175389. Available: http://legacy.library.ucsf.edu/tid/ocy56c00. Accessed 14 November 2011
132. 2000 Report P 0500/3316 Chemical analysis of mainstream smoke from cigarettes 98.fb.107, 98.fb.109, 98.fb.110, and 98.fb. 111 project juice. 06 Nov 2000. Bates number 2067592110–2067592176
133. INBIFO 1999 Report P 0500/3315 Chemical analysis of mainstream smoke from cigarettes 98.fb.107, 98.fb.113, 98.fb.114, and 98.fb. 116 project milk. 16 Dec 1999. Bates number 2067620440–2067620462. Available: http://legacy.library.ucsf.edu/tid/pnb97g00. Accessed 14 November 2011
134. INBIFO 2001 Study plan P 0500/3417 in vitro mutagenicity of total particulate matter of mainstream smoke from cigarettes with different amounts of propylene glycol in the filler salmonella typhimurium reverse mutation assay project pg Ii. 18 Sep 2001. Bates number 2067119314/2067119338. Available: http://legacy.library.ucsf.edu/tid/nly34a00. Accessed 14 November 2011
135. RoemerE 1996 Study concept chemical analyses of mainstream smoke of the standard reference cigarette 1r4f smoked various conditions. 26 Sep 1996. Bates number 2064300661/2064300663. Available: http://legacy.library.ucsf.edu/tid/awz50c00. Accessed 14 November 2011
136. RoemerE 1996 Study concept in vitro cytotoxicity (mouse embryo balb/c 3t3 cells) of mainstream smoke fractions of the standard reference cigarette 1r4f smoked under various conditions. 26 Sep 1996. Bates number 2064300659/2064300660. Available: http://legacy.library.ucsf.edu/tid/bwz50c00. Accessed 14 November 2011
137. RoemerE 1996 Study concept salmonella reverse mutation assay of total particulate matter of the standard reference cigarette 1r4f smoked under various conditions. 26 Sep 1996. Bates number 2064300657/2064300658. Available: http://legacy.library.ucsf.edu/tid/cwz50c00. Accessed 14 November 2011
138. McCannJGoldLSHornLMcGillRGraedelTE 1988 Statistical analysis of Salmonella test data and comparison to results of animal cancer tests. Mutat Res 205 183 195
139. ParodiSTaningherMRomanoPGrilliSSantiL 1990 Mutagenic and carcinogenic potency indices and their correlation. Teratog Carcinog Mutagen 10 177 197
140. BogenKT 1995 Improved prediction of carcinogenic potencies from mutagenic potencies for chemicals positive in rodents and the ames test. environmental and molecular mutagenesis 25 37 49
141. FettermanBAKimBSMargolinBHSchildcroutJSSmithMG 1997 Predicting rodent carcinogenicity from mutagenic potency measured in the Ames Salmonella assay. Environ Mol Mutagen 29 312 322
142. BakerRRMasseyEDSmithG 2004 An overview of the effects of tobacco ingredients on smoke chemistry and toxicity. Food Chem Toxicol 42 Suppl S53 83
143. BakerRRPereira da SilvaJRSmithG 2004 The effect of tobacco ingredients on smoke chemistry. Part II: casing ingredients. Food Chem Toxicol 42 Suppl S39 52
144. BakerRRPereira da SilvaJRSmithG 2004 The effect of tobacco ingredients on smoke chemistry. Part I: flavourings and additives. Food Chem Toxicol 42 Suppl S3 37
145. NeilsenKGlantzSA 2004 A tobacco industry study of airline cabin air quality: dropping inconvenient findings. Tob Control 13 Suppl 1 i20 i29
146. YanoE 2005 Japanese spousal smoking study revisited: how a tobacco industry funded paper reached erroneous conclusions. Tob Control 14 227 233; discussion 233–225
147. BarnesRLHammondSKGlantzSA 2006 The tobacco industry's role in the 16 Cities Study of secondhand tobacco smoke: do the data support the stated conclusions? Environ Health Perspect 114 1890 1897
148. BarnesRLGlantzSA 2007 Endotoxins in tobacco smoke: shifting tobacco industry positions. Nicotine Tob Res 9 995 1004
149. TongEKGlantzSA 2007 Tobacco industry efforts undermining evidence linking secondhand smoke with cardiovascular disease. Circulation 116 1845 1854
150. World Health Organization Guidelines for implementation of Article 5.3 of the WHO Framework Convention on Tobacco Control on the protection of public health policies with respect to tobacco control from commercial and other vested interests of the tobacco industry. http://www.who.int/fctc/guidelines/article_5_3.pdf. Accessed 12 April 2011
151. YergerVBMcCandlessPM 2011 Menthol sensory qualities and smoking topography: a revew of tobacco industry documents. Tobacco Control 20 ii37 ii43
152. YergerVB 2011 Menthol's potential effects on nicotine dependence: a tobacco industry perspective. Tob Control 20 ii29 ii36
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